Calcium Elevation in Mitochondria Is the Main Ca(2+) Requirement for Mitochondrial Permeability Transition Pore (mPTP) Opening

We have investigated in detail the role of intra-organelle Ca(2+) content during induction of apoptosis by the oxidant menadione while changing and monitoring the Ca(2+) load of endoplasmic reticulum (ER), mitochondria, and acidic organelles. Menadione causes production of reactive oxygen species, induction of oxidative stress, and subsequently apoptosis. In both pancreatic acinar and pancreatic tumor AR42J cells, menadione was found to induce repetitive cytosolic Ca(2+) responses because of the release of Ca(2+) from both ER and acidic stores. Ca(2+) responses to menadione were accompanied by elevation of Ca(2+) in mitochondria, mitochondrial depolarization, and mitochondrial permeability transition pore (mPTP) opening. Emptying of both the ER and acidic Ca(2+) stores did not necessarily prevent menadione-induced apoptosis. High mitochondrial Ca(2+) at the time of menadione application was the major factor determining cell fate. However, if mitochondria were prevented from loading with Ca(2+) with 10 μm RU360, then caspase-9 activation did not occur irrespective of the content of other Ca(2+) stores. These results were confirmed by ratiometric measurements of intramitochondrial Ca(2+) with pericam. We conclude that elevated Ca(2+) in mitochondria is the crucial factor in determining whether cells undergo oxidative stress-induced apoptosis.